Isolation and characterization of acyl coenzyme A carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis, which produce multiple methyl-branched mycocerosic acids.
Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin-Sepharose 4B-CL gel with d-biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 mumol min(-1) mg(-1), respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K(m) values for propionyl-CoA, ATP, Mg(2+), and NaHCO(3) were 70 muM, 100 muM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K(m) was higher and the V(max) was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively (S)-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n-fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D019735:Carbon-Carbon Ligases; D005227:Fatty Acids / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D009163:Mycobacterium bovis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*
D000214:Acyl Coenzyme A; D005227:Fatty Acids; C024343:butyryl-coenzyme A; C009061:propionyl-coenzyme A; C032992:mycocerosic acid; D000105:Acetyl Coenzyme A; D008025:Ligases; D019735:Carbon-Carbon Ligases; C018757:acyl-CoA carboxylase